Platelet-derived CXCL12 promotes arterial thrombosis. (A-B) Thrombus formation was induced by FeCl₃ in the carotid artery of ApoE^−/− mice (n = 22). The time to occlusion (A) was measured by using Doppler sonography, and thrombi were classified into “stable” and “unstable” (B) as specified in the Methods. (C) Isolated mouse blood was activated with collagen (10 µg/mL), and the concentration of CXCL12 from the releasate was determined by enzyme-linked immunosorbent assay (n = 3). (D) Tail bleeding time was assessed (n = 11). (E-I) Multiparameter analysis of thrombus formation in a collagen-coated flow chamber perfused with murine whole blood (1000 s⁻¹): platelet deposition (E), thrombus size (F), thrombus multilayer score (G), thrombus contraction score (H), and phosphatidylserine (PS) exposure (I) were assessed by Annexin V staining. (J) Representative micrographs (n = 11-15); note that Cxcl12^wtlwt mice form large and contracted thrombi, in which individual platelets are barely recognizable (closed arrow heads), whereas Cxcl12^Δplt/Δplt mice tend to generate smaller less contracted thrombi featuring clearly distinguishable individual platelets (open arrow head); scale bar overview, 50 µm; scale bar inlet, 10 µm. (K-L) Platelet activation by collagen (1, 5, and 10 µg/mL) was analyzed by upregulation of activated αIIbβ3 (K) and P-selectin (L) by flow cytometry (n = 6). Data represent mean ± standard deviation from the indicated numbers of independent experiments or mice. *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001, as analyzed by using the Mann-Whitney U test (panels A and D), Fischer’s exact test (panel B), and unpaired t tests (panels C, E-I, K, and L). MFI, mean fluorescence intensity; SAC, surface area coverage.